mRNA was isolated using RNeasy mini kit (Qiagen) and reverse transcribed by reverse transcription system (Promega) according to manufacturer’s instructions. Real time PCR was performed using an Applied Biosystem 7500 System and primers and probes for claudin-5, ZO-1 and occludin (ThermoFisher). GAPDH mRNA level was assessed in duplication for data normalization.
Tissue detection of EcoHIV/NDK was performed 1 week post infection. Tissue was harvested and processed using All Prep DNA/RNA isolation kit (Qiagen). RNA was reverse transcribed as described above. HIV detection was then conducted using the following primers and probe: NDKgag_F 5′-GACATAAGACAGGGACCAAAGG; NDKgag_R 5′-CTGGGTTTGCATTTTGGACC; NDKgag_Probe 5′-AACTCTAAGAGCCGAGCAAGCTTCAC. Normalization was conducted based on GAPDH for RNA or mouse HBB for DNA80 (link).
Tissue detection of EcoHIV/NDK was performed 1 week post infection. Tissue was harvested and processed using All Prep DNA/RNA isolation kit (Qiagen). RNA was reverse transcribed as described above. HIV detection was then conducted using the following primers and probe: NDKgag_F 5′-GACATAAGACAGGGACCAAAGG; NDKgag_R 5′-CTGGGTTTGCATTTTGGACC; NDKgag_Probe 5′-AACTCTAAGAGCCGAGCAAGCTTCAC. Normalization was conducted based on GAPDH for RNA or mouse HBB for DNA80 (link).
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